Project Summary: The proposed research leverages recent technological breakthroughs to investigate essential facets of RNA modification in the human fungal pathogen C. albicans. Increased resistance to the few treatment options available for C. albicans infection underscore the importance of understanding how fungal pathogens differ from humans at the molecular level. Our research addresses fundamental questions about essential features of C. albicans biology by investigating aspects of pseudouridylation unique to C. albicans. Our long-term objective is to understand how RNA modification and processing differ between humans and pathogenic fungi. The proposed research investigates pseudouridylation, an RNA modification found in a wide variety of RNA molecules in all kingdoms of life. Although pseudouridylation is widespread, the amount and mechanism by which pseudouridylation occurs varies tremendously between taxa. Our preliminary data suggest that a number of aspects of pseudouridylation in C. albicans are distinct from analogous processes examined in S. cerevisiae and humans. We hypothesize C. albicans pseudouridine synthases modify different classes of RNA than S. cerevisiae pseudouridine. Furthermore, we hypothesize A and B homologs of C. albicans pseudouridine synthases act on distinct sets of substrates. In addition, while C. albicans rRNA has a similar number of rRNA pseudouridylation sites as S. cerevisiae, how these modifications are catalyzed are unknown. Moreover, the role/s of C. albicans snoRNA redundancy has not been investigated. To answer these questions will demand the performance of medically relevant experiments on an important human fungal pathogen. Students will gain experience in a wide variety of fundamental molecular biology techniques including cutting-edge CRISPR genome-editing technology. Students will be directly supervised, and will participate in experimental design, data collection, interpretation, and dissemination. Furthermore, students will be put in a position to make discoveries that have a direct impact on our understanding of fungal pathogenesis.